JP7725821B2 - Method for manufacturing substrates on which nerve cells are arranged - Google Patents

Description

The present invention relates to a method for manufacturing a substrate on which nerve cells are arranged.

Nerve cells form networks in the body and function in a functionally connected state. In elucidating brain function, evaluating toxicity in nervous system diseases, and drug development, reproducing the activity of in vivo nerve cells as accurately as possible in vitro is considered important in order to increase the extrapolation of test results (correlation with clinical data).

To achieve this, it would be effective to develop a neural circuit model in which arbitrary neurons are placed in predetermined locations and functionally connected to each other through the extension of axons. However, it is difficult to precisely place neurons on a substrate using manual techniques.

Patent Document 1 (JP 2019-162097 A) attempts to position cells using a pattern of cell-adhesive and non-cell-adhesive materials. However, the inventors discovered that when cells are positioned using a pattern of cell-adhesive and non-cell-adhesive materials, neurons, in particular, tend to migrate at the cell adhesion sites, making it difficult to keep them in a desired position. Therefore, the present invention aims to provide a technology for precisely positioning neurons on a substrate while inhibiting their migration.

The method of the present invention for producing a substrate on which neurons are arranged includes the steps of: arranging, by an inkjet method, a plurality of droplets containing neurons on a substrate having an area where a cell adhesive material is arranged and an area where a cell non-adhesive material is arranged, thereby forming one or more liquid pools; and allowing the neurons in the liquid pools to settle and temporarily adhere to the substrate to form a cell aggregate, wherein the diameter of each liquid pool is 500 μm or less, and the density of neurons per liquid pool is ¹⁰⁵ cells/ ^cm2 or more.

The present invention provides a technology for precisely positioning neurons on a substrate while suppressing their migration.

FIG. 1 is a schematic diagram showing an example of an inkjet head. FIG. 2 is a schematic diagram showing an example of a waveform input to the inkjet head. FIG. 3 is a schematic diagram showing an example of a waveform input to the inkjet head. FIG. 4 is a schematic diagram showing an example of a droplet placement device. FIG. 5 is a schematic diagram showing an example of a droplet placement device. FIG. 6 is a schematic diagram showing an example of a droplet placement device. FIG. 7 is a schematic diagram showing an example of a droplet placement device. FIG. 8 is a flow chart showing an example of a method for manufacturing a neural circuit model. FIG. 9 is a schematic cross-sectional view illustrating a method for manufacturing a neural circuit model. 10(a) to (c) are micrographs taken in Experimental Example 1. FIG. 11 is a graph showing the results of Experimental Example 2. FIG. 12 is a schematic diagram illustrating a procedure for arranging a pattern of a non-cell-adhesive material and a pattern of a cell-adhesive material on a substrate and then arranging cells. 13(a) to (d) are micrographs taken in Experimental Example 3. FIG. 14 is a fluorescence microscope photograph taken in Experimental Example 4. 15(a) to 15(e) are schematic diagrams illustrating the procedure of Experimental Example 5. 16(a) to 16(d) are schematic diagrams showing the arrangement of cells in Experimental Example 6. FIG. Fig. 17(a) is a schematic diagram showing the arrangement pattern of the non-cell-adhesive material and neurons in the neural circuit model prepared in Experimental Example 7. Fig. 17(b) is a representative micrograph of the neural circuit model immediately after ejection of the cell ink in Experimental Example 7. FIG. 18 is a fluorescent micrograph of the neural circuit model prepared in Experimental Example 7. 19(a) and (b) are diagrams showing the pattern of the non-cell-adhesive material placed on the substrate in Experimental Example 8. FIG. 20(a) and (b) are fluorescence microscope photographs taken in Experimental Example 8. 21(a) and (b) are micrographs taken in Experimental Example 9.

Embodiments of the present invention will be described in detail below, with reference to the drawings where appropriate. Note that identical or corresponding parts in the drawings are designated by identical or corresponding reference numerals, and duplicate explanations will be omitted. Note that the dimensional ratios in each drawing may be exaggerated for the purpose of explanation and do not necessarily correspond to the actual dimensional ratios.

[Method for manufacturing a substrate on which nerve cells are arranged]
In one embodiment, the present invention provides a method for manufacturing a substrate on which neurons are arranged, the method comprising the steps of: arranging, by inkjet printing, a plurality of droplets containing neurons on a substrate having an area where a cell-adhesive material is arranged and an area where a non-cell-adhesive material is arranged, to form one or more liquid pools; and allowing the neurons in the liquid pools to settle and temporarily adhere to the substrate to form cell aggregates, wherein the diameter of each liquid pool is 500 μm or less and the density of neurons per pool is ¹⁰⁵ cells/ ^cm2 or more.

As described later in the Examples, the inventors have demonstrated that neuronal migration is suppressed and neurons can be precisely positioned on a substrate when the diameter of each liquid pool placed on the substrate is 500 μm or less and the density of neurons in the liquid pool is 10 ⁵ cells/cm ² or more. Furthermore, by discharging droplets containing cells onto a substrate using an inkjet method, cells can be stably positioned in units of several cells in a microscopic area on the order of micrometers.

The diameter of each reservoir may be 400 μm or less, 300 μm or less, or 200 μm or less. Reducing the diameter of the reservoir is preferable because it makes it easier to reduce the amount of expensive cells used.

Here, droplets refer to droplets ejected from an inkjet head by an inkjet method. A puddle refers to a droplet formed when multiple droplets ejected from an inkjet head land on a substrate. The diameter of a single puddle refers to the diameter of the area where a single puddle contacts the substrate. If the area where the puddle contacts the substrate is not circular, the diameter refers to a circle with the same area as the area where the puddle contacts the substrate. The density of neurons per puddle refers to the number of cells per area where a single puddle contacts the area on the substrate where the cell adhesive material is arranged .

The droplets preferably contain 1 to 50 neurons per droplet. Furthermore, the number of neurons contained per reservoir is preferably approximately 7 to 10,000. The number of neurons contained per reservoir may be 7 or more, 30 or more, or 70 or more. Furthermore, the number of neurons contained per reservoir may be 100 or less, 70 or less, or 30 or less. These upper and lower limits can be combined in any manner.

The substrate is not particularly limited as long as it can be used for cell culture. Examples of substrate materials include the organic and inorganic materials listed below. These may be used alone or in combination of two or more types.

The organic material is not particularly limited and can be selected appropriately depending on the purpose. Examples include polyethylene terephthalate (PET), polystyrene (PS), polycarbonate (PC), TAC (triacetylcellulose), polyimide (PI), nylon (Ny), low-density polyethylene (LDPE), medium-density polyethylene (MDPE), vinyl chloride, vinylidene chloride, polyphenylene sulfide, polyethersulfone, polyethylene naphthalate, polypropylene, acrylic materials such as urethane acrylate, silicone materials such as cellulose and polydimethylsiloxane (PDMS), polyvinyl alcohol (PVA), metal alginates such as calcium alginate, polyacrylamide, methylcellulose, gel-like materials such as agarose, etc.

There are no particular restrictions on the inorganic material, and it can be selected appropriately depending on the purpose. Examples include glass, ceramics, etc.

The structure of the substrate is not particularly limited as long as it can be used for cell cultivation, and examples include porous and non-porous structures. The substrate may have a porous structure, or may be a non-porous flat plate with a porous material laminated on top.

There are no limitations on the size or shape of the pores in the porous structure, and they may be, for example, a mesh structure, a textured structure, a honeycomb structure, etc. Porous structures are preferred as substrate structures because they increase the surface area on which the non-cell-adhesive material or cell-adhesive material can be fixed, and they can retain a large amount of solution, preventing drying.

If a substrate with a porous structure is used and a liquid such as a culture medium is held in the substrate beforehand, and a liquid pool is formed, the liquid is retained by the porous structure, preventing the liquid pool from drying out.

In addition, when droplets are ejected onto a dried substrate to form a liquid pool, a process for suppressing evaporation of the liquid in the liquid pool (drying suppression process) can be carried out to maintain the shape of the liquid pool and stably achieve adhesion of cells to the substrate.

Examples of drying suppression processes include (i) increasing the humidity around the liquid pool, (ii) placing a fluid that suppresses liquid evaporation on the substrate and then forming a liquid pool (here, examples of fluids that suppress liquid evaporation include oil, culture medium, buffer solution, etc.), and (iii) forming a liquid pool on the substrate and then covering the liquid pool with a fluid that suppresses liquid evaporation (e.g., oil, etc.).

When suppressing evaporation by increasing humidity, it is preferable to control humidity locally to minimize the impact on the surrounding area. Furthermore, when using oil to suppress drying, it is preferable to use oil that is biocompatible in order to minimize the impact on cells.

The manufacturing method of this embodiment may further include a step of supplying a culture medium to the substrate on which the cell aggregates have been formed. The culture medium can be appropriately selected and used to suit the cells used. Specific media include, for example, Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, D-MEM/F12, McCoy's 5A medium, Eagle's Minimum Essential Medium (EMEM), alpha Modified Eagle's Minimum Essential Medium (αMEM), and MEM (Minimum Essential Medium). Medium), RPMI1640 (Roswell Park Memorial Institute-1640) medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, Williams' Medium E, IPL41 medium, Fischer's medium, M199 medium, High Performance Medium 199, StemPro34 (Thermo Fisher Scientific), X-VIVO 10 (Chembrex), X-VIVO 15 (Chembrex), HPGM (Chembrex), StemSpan H3000 (StemCell Technologies), StemSpan SFEM (StemCell Technologies), Stemline II (Sigma-Aldrich), QBSF-60 (Quality Biological), StemPro hESC SFM (Thermo Fisher Scientific), Essential8 (registered trademark) medium (Thermo Fisher Scientific), mTeSR1 or mTeSR2 medium (StemCell Technologies), ReproFF or ReproFF2 (ReproCell), PSGro hESC/iPSC medium (System Examples of suitable medium include: NutriStem (registered trademark) medium (Biological Industries), CSTI-7 medium (Cell Science Institute), MesenPRO RS medium (Thermo Fisher Scientific), MF-Medium (registered trademark) mesenchymal stem cell growth medium (Toyobo Co., Ltd.), Sf-900II (Thermo Fisher Scientific), and Opti-Pro (Thermo Fisher Scientific).

These may be used alone or in combination of two or more. In particular, in the case of DMEM/F12 medium, it is preferable to mix DMEM medium and F12 medium in a mass ratio ranging from 6:4 to 4:6.

Additives may also be added to the culture medium. Examples of additives include those commonly used in neuronal culture, such as SM1 Supplement (Stem Cell Technologies), N2 Supplement A (Stem Cell Technologies), rat astrocyte culture supernatant (Fujifilm Wako Pure Chemical Industries), human astrocyte culture supernatant (Sciencell Research), Component N (Elixirgen Scientific), Component G2 (Elixirgen Scientific), Component P (Elixirgen Scientific), N2 Supplement (Thermo Fisher Scientific), iCell Neural Supplement B (CDI), and iCell Neuvous System Supplement, B-27. Plus (Thermo Fisher Scientific) and others.

The manufacturing method of this embodiment may further include forming multiple liquid pools in the liquid pool forming step, forming multiple cell aggregates in the leaving step, and incubating the substrate supplied with culture medium to functionally bond at least two of the cell aggregates.

When multiple liquid pools are formed on a substrate, cell aggregates are formed in each liquid pool. These cell aggregates are then supplied with culture medium and incubated, and when at least two of these cell aggregates become functionally connected, a neural circuit model can be produced. Here, functional connection between cell aggregates refers to when a neuron extends a process called an axon and forms a synaptic connection with the dendrites of another neuron. As a result, a neural circuit is formed.

Nerve cells can be broadly divided into peripheral and central nerve cells. Peripheral nerve cells include, for example, sensory nerve cells, motor nerve cells, and autonomic nerve cells. Central nerve cells include, for example, interneurons and projection neurons. Projection neurons include, for example, cortical neurons, hippocampal neurons, and amygdala neurons. Central nerve cells can also be broadly divided into excitatory neurons and inhibitory neurons. Examples include glutamatergic neurons, which are primarily responsible for excitatory transmission in the central nervous system, and GABAergic (γ-aminobutylic acid) neurons, which are primarily responsible for inhibitory transmission.

Other neurons that release neuromodulators include cholinergic neurons, dopaminergic neurons, noradrenergic neurons, serotonergic neurons, and histaminergic neurons.

In the manufacturing method of this embodiment, a cell aggregate containing cells other than neurons may be further arranged on the substrate on which neurons are arranged. The cell aggregate other than the cell aggregate containing neurons may contain cells capable of receiving a transmission signal from neurons. Examples of cells capable of receiving a transmission signal from neurons include neurons and muscle cells. Examples of muscle cells include cardiac muscle cells, skeletal muscle cells, and smooth muscle cells. One type of these cells may be used alone, or two or more types may be used in combination.

In the manufacturing method of this embodiment, the nerve cells may be primary cultured cells, passaged cells, established cell lines, immortalized cells, or nerve cells that have undergone various gene editing processes. Furthermore, from the viewpoint of making it easier to obtain a cell population containing a large number of desired nerve cells, nerve cells may be those that have been induced to differentiate from stem cells.

Examples of stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells, mesenchymal stem cells, umbilical cord blood-derived stem cells, and neural stem cells. Examples of induced pluripotent stem cells include nuclear transfer embryonic stem cells (ntES cells) and induced pluripotent stem cells (iPS cells). Examples of mesenchymal stem cells include bone marrow mesenchymal stem cells and adipose tissue-derived mesenchymal stem cells. Among these, iPS cells are preferred as stem cells.

iPS cells may be derived from healthy individuals or from patients with various nervous system disorders. They may also be genetically edited, for example, cells that have been genetically edited to contain genes that are the cause or risk factors for various nervous system disorders.

When iPS cells are derived from patients with various nervous system disorders, they can be used to construct models of those nervous system disorders. Nervous system disorders include, but are not limited to, neurodegenerative disorders, autism, epilepsy, attention-deficit hyperactivity disorder (ADHD), schizophrenia, and bipolar disorder. Neurodegenerative disorders include, for example, Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis.

The animal species from which the nerve cells are derived is not particularly limited, and examples include humans, monkeys, dogs, cattle, horses, sheep, pigs, rabbits, mice, rats, guinea pigs, and hamsters. Of these, humans are preferred.

In the manufacturing method of this embodiment, the nerve cells may be collected from a living organism, may be established and cultured, or may be differentiated from stem cells.

When producing a neural circuit model using multiple types of neurons, it is preferable that one cell assembly contains one type of neuron. Furthermore, each of the multiple cell assemblies may contain different types of neuron depending on the purpose.

The following describes the process of placing droplets containing neurons on a substrate to form one or more liquid pools. This process is carried out by ejecting a cell suspension (cell ink) containing at least neurons and a cell drying inhibitor as droplets using an inkjet system.

Inkjet droplet ejection methods include, for example, the so-called piezo method, which uses a piezoelectric element as a pressure generating means to pressurize the cell ink and change the volume of the cell suspension to eject droplets (see, for example, Japanese Patent Publication No. 2-51734), the so-called thermal method, which uses a heating resistor to heat the cell ink and generate bubbles (see, for example, Japanese Patent Publication No. 61-59911), and the electrostatic method, which arranges a vibrating plate and an electrode opposite each other and changes the volume of the cell ink to eject droplets by deforming the vibrating plate using electrostatic force generated between the vibrating plate and the electrode (see, for example, Japanese Patent Publication No. 6-71882).

(inkjet head)
A specific embodiment of an inkjet head used to eject droplets of cell ink containing nerve cells and a cell drying inhibitor will be described below. Fig. 1 is a schematic diagram showing an example of an inkjet head. In Fig. 1, a piezoelectric element is used as a pressure generating means. Figs. 2 and 3 are schematic diagrams showing examples of input waveforms to the inkjet head.

The droplet ejection head 10 has a liquid chamber 12 that holds cell ink 11, a nozzle 15, a membrane 13 that is a film-like member, a vibration unit 16 that imparts vibrations to the membrane 13, and a drive unit 14 that applies a voltage to the vibration unit 16 as a specific drive signal to vibrate the vibration unit 16.

The liquid chamber 12 is provided with an air vent 17 for opening the liquid chamber to the atmosphere. The droplet ejection head 10 applies vibrations to the cell ink, causing droplets of the cell ink to be ejected from the nozzles.

The driver 14 is capable of applying an ejection waveform Pj as a drive signal to the vibration generator 16, thereby controlling the vibration state of the membrane 13 and ejecting the cell-cell ink 11 held in the liquid chamber 12 in the form of droplets. The ejection waveform Pj is preferably set to a drive signal that includes the membrane 13's natural vibration period To in order to resonate the membrane 13 and eject the cell-cell ink 11 with less voltage. The ejection waveform Pj can be not only a triangular wave or a sine wave, but also a triangular wave with gentler edges obtained by applying a low-pass filter. Furthermore, the driver 14 is capable of applying a vibration-isolating waveform Ps as a drive signal to the vibration generator 16, which suppresses residual membrane vibration after droplet ejection. This quickly suppresses residual membrane vibration after droplet formation, enabling higher-frequency continuous ejection. Furthermore, the reduction in satellites and mist allows for more precise control of droplet volume. The vibration isolation waveform Ps can be not only a triangular wave or a sine wave, but also a triangular wave with gentler edges that has been filtered through a low-pass filter.

The amount of cell ink 11 that can be held in the liquid chamber 12 is not particularly limited, and it is possible to hold, for example, approximately 1 μL to 1 mL of liquid. In particular, when using an expensive liquid such as cell ink with dispersed cells, it is preferable that droplets can be formed with a small amount of liquid, and a configuration that can hold approximately 1 μL to 50 μL of liquid is desirable.

The shape of the membrane 13 may be circular, elliptical, or rectangular. There are no particular restrictions on the material of the membrane 13, but if it is too soft, the membrane will vibrate easily, making it difficult to immediately stop the vibration when droplets are not being ejected. Therefore, it is preferable that the material has a certain degree of hardness. Examples of materials that can be used for the membrane 13 include metal materials, ceramic materials, and polymeric materials with a certain degree of hardness.

The nozzle 15 is preferably formed as a substantially circular through-hole in the center of the membrane 13. The vibrator 16 can be, for example, a piezoelectric element. By applying a voltage, compressive stress is applied in the lateral direction of the page, causing the membrane 13 to deform. The piezoelectric element can be made of the commonly used lead zirconate titanate, for example. Other piezoelectric materials that can be used include bismuth iron oxide, metal niobate, barium titanate, or any of these materials to which a metal or a different oxide has been added.

The means for applying vibrations to membrane 13 to deform it is not limited to piezoelectric elements. For example, a material with a different linear expansion coefficient from that of the membrane can be attached to membrane 13, and by heating it, membrane 13 can be deformed by taking advantage of the difference in linear expansion coefficients. In this case, for example, a heater is formed on the material with the different linear expansion coefficient, and when electricity is applied, the heater heats up, causing membrane 13 to deform.

(Cell Ink)
Next, we will explain the cell ink. The cell ink contains at least neurons and a cell drying inhibitor. Furthermore, the cell suspension (cell ink) contains a dispersion medium to disperse the cells, and may contain other additives such as dispersants and pH adjusters as necessary. The neurons are the same as those described above.

A cell desiccation inhibitor is a substance that coats the surface of cells and prevents them from drying out. Examples include polyhydric alcohols, gel-like polysaccharides, and proteins selected from the extracellular matrix.

There are no particular limitations on the polyhydric alcohol as long as it does not damage cells, and examples include glycerin, diglycerin, diethylene glycol, 1,3-butanediol, 1,2,3-butanetriol, 1,2,4-butanetriol, triethylene glycol, tetraethylene glycol, propylene glycol, and polyethylene glycol. These may be used alone or in combination of two or more. Of these, glycerin is preferred. Glycerin has low toxicity to cells and can be expected to suppress drying even when added in small amounts.

Gel-like polysaccharides are polysaccharides in a gel state. There are no particular limitations on gel-like polysaccharides and they can be selected appropriately depending on the purpose. Examples include calcium alginate, gellan gum, agarose, guar gum, xanthan gum, carrageenan, pectin, locust bean gum, tamarind gum, diutan gum, and carboxymethylcellulose. These may be used alone or in combination of two or more. Among these, calcium alginate is preferred. Calcium alginate is a salt in which calcium ions are bound to the carboxyl groups of alginic acid. Because calcium ions are divalent, they bind across the two carboxyl groups (ionic cross-linking), thickening the ink and preventing it from drying out. The calcium ions are contained in the dispersion medium and are thought to bind to excess calcium ions that become concentrated during drying, potentially regulating osmotic pressure.

The protein selected from the extracellular matrix is not particularly limited and can be selected appropriately depending on the purpose. Examples include collagen, laminin, fibronectin, elastin, fibrin, etc. These may be used alone or in combination of two or more. Of these, collagen is preferred. There are many different types of collagen, but it is known to thicken depending on the concentration and temperature. By including it in the cell ink, the viscosity can be increased as the concentration increases.

Preferably, the dispersion medium is a cell culture medium or buffer solution. The medium is the same as described above. The buffer solution is used to adjust the pH, and any known buffer solution can be selected and used as appropriate.

Because the liquid pools placed on the substrate are tiny, evaporation of the liquid in the pools occurs within tens of seconds to a few minutes, causing a rapid rise in the concentration of components in the pools. This change in concentration causes sudden changes in osmotic pressure, etc., which can damage the cells in the pools and, in the worst case, lead to cell death. Therefore, it is preferable to suppress evaporation of the liquid in the pools using the following methods.

For example, if a substrate with a porous structure is used and a liquid such as a culture medium is held in the substrate beforehand, and a liquid pool is formed, the liquid is held in place by the porous structure, preventing the liquid pool from drying out.

Furthermore, when droplets are ejected onto a dried substrate to form a liquid pool, a step of suppressing evaporation of the liquid in the liquid pool (drying suppression step) can be carried out to maintain the shape of the liquid pool and stably achieve adhesion of cells to the substrate. Examples of the drying suppression step include steps similar to those described above.

(Step of disposing droplets)
Next, we will explain the process of placing one or more droplets containing cells on a substrate to form one or more puddles. In the droplet placement process, droplets of cell ink are ejected at target positions on the substrate. Droplets containing nerve cells may be placed at one location on the substrate, or at multiple locations on the substrate. Multiple droplets of cell ink ejected from an inkjet head are placed at one location on the substrate, forming one puddle.

By adjusting the timing of droplet ejection, it is possible to adjust the position of cells adhering to the area where the cell adhesive material is placed (cell adhesion area). Furthermore, by adjusting the amount of cell ink ejected (number of droplets and droplet volume) and the cell concentration, it is possible to adjust the number of cells placed on the substrate.

Cells in a liquid pool placed on a substrate settle and temporarily adhere to the substrate, forming a cell aggregate. With the inkjet method, the droplets of cell ink are much smaller than with manual techniques, so the time it takes for cells to temporarily adhere to the substrate is extremely short. Furthermore, by placing droplets using the inkjet method, neurons are placed in predetermined positions, resulting in a high degree of accuracy in the shape of the cell pattern. There is a high probability that a predetermined number of neurons can be placed in a predetermined position, or that the number of placed cells is the desired number, and the accuracy in placing the desired number of neurons is high. Furthermore, the survival rate of placed neurons after a predetermined time has passed is also high.

(Droplet placement device)
Specific embodiments of an apparatus for disposing droplets on a substrate (hereinafter, sometimes referred to as a "droplet disposing apparatus") will be described below, but the present invention is not limited to these embodiments.

The droplet placement device has a stage unit and an inkjet head, which is a droplet ejection means for ejecting droplets of cell ink. The stage unit holds the substrate. The droplet ejection head has the same configuration as the inkjet head described above.

Figures 4 to 7 show an example of a droplet placement device equipped with an inkjet head. The droplet placement device 400 shown in Figure 4 has a stage unit 31 and an inkjet head 21. As described above, the inkjet head 21 has a liquid chamber unit 25, a vibration unit 27, a drive unit 26, and a membrane 28. The inkjet head 21 also has an atmosphere vent unit 24.

The droplet placement device may be configured to eject droplets of not only cell ink but also a solution containing a non-cell-adhesive material or a cell-adhesive material. The droplet placement device 500 shown in Figure 5 includes an inkjet head 21 that ejects droplets of cell ink, as well as an inkjet head 23 that ejects droplets of a solution containing a non-cell-adhesive material or a cell-adhesive material. The basic configuration of the inkjet head 23 is the same as that of the inkjet head 21. In Figure 5, reference numeral 29 denotes a drive unit, and reference numeral 30 denotes a liquid chamber that holds a solution of the non-cell-adhesive material or the cell-adhesive material. Like the inkjet head 21, the inkjet head 23 also has a vibration unit and a membrane (not shown).

The droplet placement device can also place droplets containing two or more types of cells. The droplet placement device 600 shown in Figure 6 can be equipped with multiple inkjet heads 21 that eject droplets of cell ink. Figure 6 shows an example equipped with inkjet head 21 and inkjet head 22, which has the same configuration as inkjet head 21. Figure 7 shows a droplet placement device 700 equipped with inkjet head 23 in addition to inkjet heads 21 and 22. In addition to the configuration described above, the droplet placement device may also be equipped with a holding unit that holds the inkjet heads, a mechanism that controls the relative position of the stage and inkjet head, etc.

The substrate on which neurons are arranged has an area where a cell adhesive material is arranged and an area where a non-cell adhesive material is arranged. In this specification, the area where a cell adhesive material is arranged may be referred to as a cell adhesive portion. Furthermore, the area where a non-cell adhesive material is arranged may be referred to as a non-cell adhesive portion. As will be described later in the Examples, by arranging a pattern of non-cell adhesive material on the surface, it becomes possible to more flexibly control axonal outgrowth when neurons are arranged.

By disposing a non-cell-adhesive material on the substrate, it is possible to regulate the direction of cell growth. Furthermore, by disposing a non-cell-adhesive material on the substrate, it is possible to dispose multiple types of cells without them intermixing with each other. When disposing multiple types of cells using the inkjet method, it is preferable to provide one inkjet head for ejecting droplets of cell ink for each type of cell, in order to avoid contamination between the cells.

The pattern of the non-cell-adhesive material has areas where the non-cell-adhesive material is disposed and areas where the non-cell-adhesive material is not disposed, and the areas where the non-cell-adhesive material is not disposed may have a linear shape (line shape). In this case, it is preferable that the width of the linear shape is 100 μm or less.

When the width of the linear shape is 100 μm or less, axons can extend along the linear shape where no non-cell-adhesive material is arranged. In other words, the area sandwiched between two linear patterns of non-cell-adhesive material can be used as a path for axon extension.

The substrate on which cells are arranged not only has a pattern of a non-cell-adhesive material arranged on its surface, but also has a pattern of a cell-adhesive material arranged on its surface. The cell-ink droplets may be arranged in contact with the cell-adhesive material. When the cell-ink droplets are arranged in contact with the cell-adhesive material and the area of the cell-adhesive material pattern is smaller than the area of the cell-ink puddle arranged on the substrate in contact with the substrate, the cells contained in the droplets tend to settle and temporarily adhere to the substrate, and then migrate and aggregate on the cell-adhesive material pattern. Therefore, by arranging a pattern of cell-adhesive material on the substrate, it is possible to control the arrangement of cells.

(Cell non-adhesive material)
Examples of non-cell adhesive materials include polydimethylsiloxane (PDMS), gels of metal alginates (such as calcium alginate), polyhydroxyethyl methacrylate (pHEMA), polyethylene glycol (PEG), and derivatives thereof.

Here, we will explain the case where the non-cell-adhesive material is polyethylene glycol. In this case, for example, by contacting a first solution containing a hyperbranched polymer with a polyethylene glycol backbone and one or more nucleophilic or electrophilic functional groups on its side chains and/or ends with a second solution containing a hyperbranched polymer with a polyethylene glycol backbone and one or more nucleophilic or electrophilic functional groups on its side chains and/or ends, they can be crosslinked to form a pattern of the non-cell-adhesive material.

A "polyethylene glycol-based hyperbranched polymer" (hereinafter sometimes simply referred to as a "hyperbranched polymer" or "multi-arm PEG") is a polymer used as a gelling material. Two types of multi-arm PEGs, each with a nucleophilic functional group and an electrophilic functional group at the ends of multiple polyethylene glycol (PEG) branches, can crosslink with each other to form a gel with a mesh-like network (multi-arm PEG gel).

For example, in the case of two types of four-branched polymers (hereinafter sometimes referred to as "Tetra-PEG") that have a nucleophilic functional group and an electrophilic functional group at the ends of each of the four PEG branches, a gel called a "Tetra-PEG gel" can be formed, which has a uniform network structure.

There are no particular limitations on the number of branches in a hyperbranched polymer. Generally, any PEG with three or more branches, including electrophilic and nucleophilic ends, can be used without any problems, and can be selected appropriately as needed. As long as each PEG has a nucleophilic functional group and an electrophilic functional group, the two or more PEGs that make up the multi-arm PEG may have different numbers of branches. Among multi-arm PEGs, tetra-PEG gel is known to have an ideal uniform network structure.

Furthermore, Tetra-PEG gel can be easily and quickly formed on-site by simply mixing the two types of Tetra-PEG contained in the first and second solutions, and the gelation time can be controlled by adjusting the pH and concentration of each Tetra-PEG. Additionally, because PEG is the main component, it has excellent biocompatibility. By ejecting a solution containing cells and Tetra-PEG using a droplet ejection device and reacting the two types of Tetra-PEG to form a Tetra-PEG gel, it becomes possible to position cells in three dimensions.

In one embodiment, Tetra-PEG may be a compound having a structure represented by the following general formula (1):

The m's in the formula (1) may be the same or different. The closer the m's are, the more uniform the three-dimensional structure can be, and the stronger the gel will be. Therefore, to obtain a high-strength gel, it is preferable that the m's are the same. If the m's are too high, the gel strength will be weak, and if the m's are too low, the gel will be difficult to form due to steric hindrance of the compound. Therefore, each m' may be an integer value of 25 to 250, preferably 35 to 180, more preferably 50 to 115, and particularly preferably 50 to 60. The molecular weight thereof may be 5 x 10 ³ to 5 x 10 ⁴ Da, preferably 7.5 x 10 ³ to 3 x 10 ⁴ Da, and more preferably 1 x 10 ⁴ to 2 x 10 ⁴ Da.

In the above formula (1), ^X1 is a linker moiety connecting the functional group and the core portion. Each ^X1 may be the same or different, but is preferably the same in order to produce a high-strength gel having a uniform three-dimensional structure. ^X1 represents a _C1 - _C7 alkylene group, a _C2 - _C7 alkenylene group, -NH-Ra-, -CO-Ra-, -Rb-O-Rc-, -Rb-NH-Rc-, -Rb-CO2- _Rc- , -Rb-CO2- _NH -Rc-, -Rb-CO-Rc-, or -Rb-CO-NH-Rc-. Here, Ra represents a _C1 - _C7 alkylene group, Rb represents a _C1 - _C3 alkylene group, and Rc represents a _C1 - _C5 alkylene group.

The term "C ₁ -C ₇ alkylene group" refers to an alkylene group having from 1 to 7 carbon atoms which may have a branch, and includes a linear C ₁ -C ₇ alkylene group, or a C ₂ -C ₇ alkylene group having one or more branches (having from 2 to 7 carbon atoms including the branches). Examples of C ₁ -C ₇ alkylene groups include a methylene group, an ethylene group, a propylene group, and a butylene group. Examples of C ₁ -C ₇ alkylene groups include -CH ₂ -, -(CH ₂ ) ₂ -, -(CH ₂ ) ₃ -, -CH(CH ₃ )-, -(CH(CH ₃ )) ₂ -, -(CH ₂ ) ₂ -CH(CH ₃ )-, -(CH ₂ ) ₃ -CH(CH ₃ )-, -(CH ₂ ) ₂ -CH(C ₂ H ₅ )-, -(CH ₂ ) ₆ -, -(CH ₂ ) ₂ -C(C ₂ H ₅ ) ₂ -, and -(CH ₂ ) ₃ C(CH ₃ ) ₂ CH ₂ -.

A "C ₂ -C ₇ alkenylene group" is a straight-chain or branched alkenylene group having 2 to 7 carbon atoms and one or more double bonds in the chain. For example, it may be a divalent group having a double bond formed by removing 2 to 5 hydrogen atoms from adjacent carbon atoms of an alkylene group.

In the above formula (1), ^Y1 is a functional group for forming a meshwork network by a cross-end coupling reaction, which is a crosslinking reaction by a covalent bond, as described above, and is selected from nucleophilic functional groups or electrophilic functional groups.

The "nucleophilic functional group" is not limited, but a thiol group is preferred, for example, from the viewpoint of shortening the gelation time. The functional groups may be the same or different, but it is preferable that they are the same. Having the same functional groups ensures uniform reactivity with the nucleophilic functional group that is the target of the crosslinking reaction, making it easier to obtain a high-strength gel with a uniform three-dimensional structure. Hereinafter, Tetra-PEG having a nucleophilic functional group may be referred to as "nucleophilic Tetra-PEG."

The "electrophilic functional group" is not limited, but a maleimidyl group is preferred, for example, from the viewpoint of shortening the gelation time. The functional groups may be the same or different, but it is preferable that they are the same. Having the same functional groups ensures uniform reactivity with the nucleophilic functional groups that are the target of the crosslinking reaction, making it easier to obtain a high-strength gel with a uniform three-dimensional structure. Hereinafter, Tetra-PEG having an electrophilic functional group may be referred to as "electrophilic Tetra-PEG."

Nucleophilic Tetra-PEG and electrophilic Tetra-PEG can be mixed so that the molar ratio of nucleophilic functional groups to electrophilic functional groups is 0.5:1 to 1.5:1. Because these functional groups can react and crosslink at a 1:1 ratio, a mixing molar ratio closer to 1:1 is preferable, but to form a high-strength hydrogel, a ratio of 0.8:1 to 1.2:1 is preferred. Furthermore, when the pH of the dispersion medium in the first or second solution is 5 to 10, the concentration of Tetra-PEG contained in each solution should be in the range of 0.3 to 20%, and when the pH is 6 to 10, a range of 1.7 to 20% is preferred.

The first solution may be composed of either nucleophilic Tetra-PEG or electrophilic Tetra-PEG, and the second solution may be composed of the other Tetra-PEG.

First Solution
The "first solution" is an aqueous solution containing, as components, a multi-branched polymer having a skeleton of either polyethylene glycol having one or more nucleophilic functional groups at least on the side chains and/or ends or polyethylene glycol having one or more electrophilic functional groups, and a dispersion medium, and may contain cells or cell-active additives as necessary.

Multiple types of first solutions may be present. In this case, the hyperbranched polymer, dispersion medium, cells, and cell-interacting additives each containing a skeleton of either polyethylene glycol having one or more nucleophilic functional groups or polyethylene glycol having one or more electrophilic functional groups at the side chain and/or end may be different in whole or in part from each other. For example, the dispersion medium contained in first solution a and the dispersion medium contained in first solution b may be the same or different. When multiple types of first solutions are present, for example, when forming a layered hydrogel, the dispersion medium can be changed as desired in each layer.

Second Solution
The "second solution" is an aqueous solution containing, as essential components, a multi-branched polymer having a polyethylene glycol backbone and one or more functional groups (nucleophilic functional groups or electrophilic functional groups) at its side chains and/or ends that are different from those in the first solution, and a dispersion medium, and may contain cells or cell-active additives as necessary.

There may be multiple types of second solutions. In this case, the hyperbranched polymer with a different functional group from that of the first solution, which has a polyethylene glycol skeleton having one or more nucleophilic or electrophilic functional groups at its side chains and/or ends, the dispersion medium, the cells, and the cell-interacting additives contained in each second solution may be different in whole or in part from those contained in the first solution. For example, the cells contained in second solution a and the cells contained in second solution b may be the same or different.

<Gel formation process>
The "gel formation process" is a process in which the first solution and the second solution are mixed together, causing a reaction between the hyperbranched polymers having a polyethylene glycol skeleton contained in each solution, thereby forming a hydrogel. Either the first solution or the second solution may be ejected using a droplet ejection device, or both the first solution and the second solution may be ejected using a droplet ejection device.

A "droplet ejection device" is a means of ejecting droplets of a solution stored in a liquid chamber, causing them to land on a target site. Examples of ejection methods used by ejection devices include the inkjet method and the gel extrusion dispenser method. With the inkjet method, the solution is ejected from an ejection hole (nozzle). The ejection method is capable of ejecting minute amounts of solution (sometimes called droplets) from an ejection hole, making it possible to create highly accurate three-dimensional structures.

The volume of the ejected droplets can be any volume. Preferably, it is 9 pL or more, 15 pL or more, 20 pL or more, 30 pL or more, 40 pL or more, 50 pL or more, 60 pL or more, 70 pL or more, 80 pL or more, 90 pL or more, or 100 pL or more, and 900 pL or less, 800 pL or less, 700 pL or less, 600 pL or less, 500 pL or less, 400 pL or less, or 300 pL or less.

"Depositing" refers to bringing a solution into contact with a target site. This is achieved by ejecting droplets onto the target site using an ejection method. There are no particular restrictions on the location where the solution is ejected, as long as the solution is ejected so that it lands on the desired target site. Furthermore, the respective landing sites may be separated, or may be partially in contact or overlapping.

Gel formation occurs through a reaction between a hyperbranched polymer with a polyethylene glycol skeleton having nucleophilic and electrophilic functional groups, and therefore, a hydrogel is formed when the second solution lands on the first solution. When the second solution is ejected using a droplet ejection device, a single impact typically results in the formation of a dot-shaped hydrogel, although this is not limited to this. In this specification, "dot (shape)" refers to a point-like shape. Therefore, it is not limited to a perfect circle or hemisphere, and may include any shape, such as an approximately circular or approximately hemispherical shape, a polygonal shape, an irregular shape, or a combination thereof. Furthermore, the dot (shape) only needs to have a predetermined length and thickness in terms of its three-dimensional structure. When the second solution is ejected multiple times, a variety of hydrogel shapes, with dot-shaped hydrogels being the smallest unit, are formed.

The volume of the hydrogel formed by a crosslinking reaction resulting from a single impact, i.e., the dot-shaped hydrogel, depends on the number of times the second solution is dispensed onto the same location. Furthermore, the larger the nozzle diameter and the more times the solution is dispensed onto the same location, the larger the volume of the dot-shaped hydrogel. Therefore, the volume of the dot-shaped hydrogel can be adjusted by changing the number of times the solution is dispensed onto the same location and the nozzle diameter. While not limited to this, the volume of the dot-shaped hydrogel herein is 9 pL or more, 15 pL or more, 20 pL or more, 30 pL or more, 40 pL or more, 50 pL or more, 60 pL or more, 70 pL or more, 80 pL or more, 90 pL or more, or 100 pL or more, with 900 pL or less, 800 pL or less, 700 pL or less, 600 pL or less, 500 pL or less, 400 pL or less, and 300 pL or less being preferred. The diameter of the dot-shaped hydrogel is not limited, but should be in the range of 10 μm to 300 μm, and the thickness should be in the range of 5 μm to 150 μm.

Since the second solution is ejected any number of times, multiple dot-shaped hydrogels may be formed. The dot-shaped hydrogels may be in contact with each other in whole or in part. By arranging multiple dot-shaped hydrogels in a series, it is possible to form hydrogels of any shape, not just dots. The shape can be selected appropriately depending on the purpose. For example, by arranging dots in one axial direction, it is possible to form linear hydrogels. Furthermore, by arranging linear hydrogels in the same plane without any gaps, it is possible to form a film-like (planar) hydrogel.

《Removal process》
The "removal step" is a step of removing the unreacted, i.e., uncrosslinked, hyperbranched polymer present on the substrate or on the hydrogel after the gel formation step. This step is an optional step and may be performed as needed.

The removal method is not particularly limited, and any known removal method can be used as long as it does not have a physical, biological, or chemical effect on the formed hydrogel. A commonly used and simple removal method is to wash the support or hydrogel with an appropriate cleaning solution.

There are no particular restrictions on the cleaning solution used in the cleaning method, as long as it does not affect the hydrogel or cells. It can be selected appropriately taking into consideration factors such as pH and osmotic pressure. A buffer solution or culture medium is preferred.

The cleaning method may involve pouring a cleaning solution over the support or hydrogel, or immersing the support or hydrogel in the cleaning solution to reduce physical damage to the hydrogel. Cleaning may be performed multiple times in a single step.

(Cell adhesive material)
Examples of the cell adhesive material include proteins selected from the extracellular matrix. Proteins selected from the extracellular matrix are the same as those described above. The cell adhesive material may be discharged using a droplet discharge device and placed on the substrate. The droplet discharge device is the same as that described above.

[Neural circuit model]
In one embodiment, the present invention provides a neural circuit model comprising a substrate and a plurality of cell assemblies discretely arranged on the substrate, each of the plurality of cell assemblies including a neuron, and at least two of the cell assemblies being functionally connected. In the neural circuit model of this embodiment, each of the cell assemblies preferably includes 7 to 10,000 cells.

The neural circuit model of this embodiment can accurately reproduce the activity of neurons in vivo in vitro, and can be used to elucidate brain function, evaluate toxicity to nervous system diseases, and develop new drugs.

In the neural circuit model of this embodiment, the substrate and neurons are the same as those described above. Furthermore, functional connection between cell assemblies is the same as that described above, and refers to a neuron extending a process called an axon and forming a synaptic connection with the dendrites of another neuron. As a result, a neural circuit is formed.

In the neural circuit model of this embodiment, the substrate may have a pattern of a non-cell-adhesive material arranged on its surface. Alternatively, the substrate may have a pattern of a cell-adhesive material arranged on its surface.

The arrangement pattern and number of cell aggregates on the substrate are not particularly limited, and may be, for example, circular, rectangular, or lattice-shaped. Furthermore, the functional bonds between cell aggregates arranged on the substrate may be random, or may be bonded so that each cell aggregate is circulated.

[Method of manufacturing a neural circuit model]
In one embodiment, the present invention provides a method for producing a neural circuit model, comprising: a cell culture carrier formation step of arranging a cell-adhesive material and a cell-non-adhesive material on a substrate to form a microstructure for cell arrangement; a cell arrangement step of arranging neurons on the substrate; a step of leaving the arranged neurons to settle and temporarily adhere to the substrate to form a cell aggregate; a medium addition step of supplying medium to the substrate on which the cell aggregate has formed; and a culture step of culturing the cell aggregate to form a neural circuit model.

In the manufacturing method of this embodiment, the substrate may be one of those described above, or may be a non-porous flat plate member with a porous member laminated on it. Examples of non-porous flat plate members include those made from the substrate materials described above, and more specifically, examples include culture dishes, glass, etc.

The porous member may be, for example, a porous membrane. The pore size of the porous member is preferably large enough to prevent cells from becoming embedded therein, for example, a pore size of 1 μm or less.

Figure 8 is a flow diagram of the manufacturing method of this embodiment. As shown in Figure 8, first, in the cell culture carrier formation process, a cell adhesive material and a cell non-adhesive material are placed on a substrate to form a microstructure for cell placement. The cell adhesive material and the cell non-adhesive material are the same as those described above.

Next, in the cell placement process, cells are placed in the cell placement region of the cell placement microstructure. Cell placement is preferably performed by an inkjet method. Furthermore, if multiple types of cells are to be placed in the cell placement microstructure, all types of cells are placed.

In the manufacturing method of this embodiment, it is preferable to perform a drying prevention treatment. For example, if a substrate with a porous structure is used and a liquid such as a culture medium is held in the substrate beforehand, and then a liquid pool is formed, the liquid is held in the porous structure, preventing the liquid pool from drying out.

Furthermore, when droplets are ejected onto a dried substrate to form a liquid pool, a process for suppressing evaporation of the liquid in the liquid pool (drying suppression process) can be carried out to maintain the shape of the liquid pool and stably achieve adhesion of cells to the substrate. Examples of the drying suppression process include the same processes as those described above. The drying suppression treatment may be carried out after the cell placement process or before the cell culture carrier formation process.

Next, in the leaving step, the cells are left to stand until all the cells have adhered. Next, in the medium addition step, medium is added. The medium is the same as that described above. Next, in the culture step, the cell aggregates are cultured to form a neural circuit model.

Figure 9 is a schematic cross-sectional view illustrating a method for manufacturing a neural circuit model. As shown in Figure 9, the cell culture support 930 is made by laminating a porous member 920 on a flat plate member 910.

The method for forming a microstructure for cell placement on a porous member is not particularly limited, but in the example shown in Figure 9, the porous member 920 is immersed in the first solution 940 described above, then placed on a flat member 910, and the second solution 950 is ejected from an inkjet head.

When the first solution 940 and the second solution 950 react, a hydrogel (Tetra-PEG gel) 960, a cell non-adhesive material, is formed. The reaction between the first solution 940 and the second solution 950 can be carried out, for example, by leaving the mixture to stand for 30 minutes in a high-humidity environment. Here, a high-humidity environment with a relative humidity of 90% or higher is preferable.

Next, the cells are placed. In the example of Figure 9, the cells 980 are placed by ejecting the above-mentioned cell ink 970 using the inkjet method. At this time, the culture medium is held in the porous member 920, which reduces the impact when the ejected microdroplets of cell ink 970 land, thereby reducing damage to the cells. Furthermore, the culture medium is held in the porous member 920, which prevents the microdroplets from drying out.

The present invention will now be described in more detail using examples, but the present invention is not limited to the following examples.

[Experimental Example 1]
(Evaluation of cell migration 1)
Cells were seeded on the substrate and migration was evaluated. A glass slide coated with Matrigel (registered trademark, Corning) was used as the substrate. PC12 cells, a cell line derived from rat pheochromocytoma, were used. Cell seeding was performed using the inkjet method.

Preparation of cell ink
First, the cells were stained by dissolving a green fluorescent dye (trade name: Cell Tracker Green, manufactured by Thermo Fisher Scientific) in dimethyl sulfoxide (DMSO) at a concentration of 10 mmol/L (mM) and mixing it with the medium to prepare a medium containing the green fluorescent dye at a concentration of 10 μmol/L (μM).

Next, 5 mL of serum-free medium containing green fluorescent dye was added to the dish of cultured PC12 cells, and the cells were cultured for 30 minutes in an incubator (KM-CC17RU2, Panasonic Corporation, 37°C, 5% CO2 by _volume environment). The cells were then detached from the dish by trypsinization to obtain a cell suspension. A portion of the cell suspension was then placed on a PMMA plastic slide, and the cell number was counted using a Countess Automated Cell Counter (Thermo Fisher Scientific).

The dispersion medium for the cell ink was PBS(-) supplemented with 0.5% by mass of glycerin (molecular biology grade, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) as a cell drying inhibitor. PC12 cells were dispersed in the dispersion medium to a concentration of 6 x ¹⁰ cells/mL to obtain the cell ink.

<Cell ejection>
The liquid chamber of the cell ejection head of the device shown in Figure 4 was filled with cell ink. Subsequently, droplets of the cell ink were ejected onto the substrate, forming pools. The diameter of each pool was approximately 200 μm. Each pool on the substrate contained approximately 100 cells, giving a cell density of 3 x ¹⁰ cells/ ^cm2 . The cell density in a pool refers to the number of cells per area where the pool on the substrate is in contact with the substrate.

Figure 10(a) is a micrograph taken immediately after the cell ink was ejected. After the droplets of cell ink had landed, they were left in a high-humidity environment of 95% or higher relative humidity for approximately 10 minutes, during which time the cells within the droplets settled and temporarily adhered to the substrate, forming cell aggregates. Next, culture medium was gently added. Figure 10(b) is a micrograph taken immediately after the culture medium was added.

Cell culture
The cells were then cultured for one day in an incubator at 37°C and 5% _CO2 by volume. Figure 10(c) shows a fluorescence microscope image of Cell Tracker Green fluorescence observed in the cells one day after the start of culture. The results revealed that the cells migrated and moved from their positions immediately after ejection. When this migration occurs, it becomes difficult to keep the cells in place.

[Experimental Example 2]
(Evaluation of cell migration 2)
The number of cells in the pool was varied and cells were seeded onto the substrate to evaluate the occurrence of migration. A slide glass with a surface coated with Matrigel (registered trademark) was used as the substrate. PC12 cells were used. The cells were seeded using the inkjet method.

The cell ink was prepared and the cells were ejected in the same manner as in Experimental Example 1. However, the number of cells in the puddle was varied when ejecting the cells. Specifically, one to three cells (cell density: 2 x ¹⁰⁴ cells/ ^cm2 ), three to six cells (cell density: 6 x ¹⁰⁴ cells/ ^cm2 ), and seven to eleven cells (cell density: 1 x ¹⁰⁵ cells/ ^cm2 ) were placed per puddle on the substrate. Here, cell density refers to the number of cells per area where the puddle placed on the substrate contacts the substrate. The diameter of each puddle was 100 μm.

The cells were then cultured for one day in an incubator at 37°C and 5% _CO2 by volume. Individual cells were observed over time using an inverted microscope (model "CKX41," manufactured by Olympus Corporation) to measure the migration distance of each cell. Figure 11 is a graph showing the results of measuring the migration distance of cells one day after the start of culture. The vertical axis of the graph represents the migration distance (μm).

As a result, it was found that cell migration was suppressed when the number of cells per reservoir was 7 or more and the cell density in the reservoir was 10 ⁵ cells/cm ² or more.

[Experimental Example 3]
(neuron pattern control)
A pattern of a non-cell-adhesive material and a pattern of a cell-adhesive material were placed on a substrate. The substrate was a glass slide on which a 13 mm diameter polyester porous culture membrane (trade name: ipCELLCULTURE Track Etched Membrane, pore size: 0.45 μm, pore density: 4 × 10 ⁶ cells/cm ² , thickness: 12 μm, manufactured by it4ip) was laminated.

Next, neurons were seeded onto this substrate, and axonal outgrowth was observed. Human iPS cell-derived GABAergic neurons (Elixirgen Scientific) were used as the neurons. Figure 12 is a schematic diagram illustrating the procedure for placing patterns of non-cell-adhesive material and cell-adhesive material on the substrate, and then placing cells.

Preparation of First Solution
Tetra-PEG-SH (trade name "SUNBRIGHT PTE-100SH", manufactured by Yuka Sangyo Co., Ltd.) was dissolved in PBS(-) and then filtered through a filter having an average pore size of 0.2 μm (trade name "Minisart Syringe Filter 175497K", manufactured by Sartorius) to obtain a first solution containing 2% Tetra-PEG-SH.

Preparation of second solution
Tetra-PEG-maleimidyl (trade name "SUNBRIGHT PTE-100MA", manufactured by Yuka Sangyo Co., Ltd.) and Matrigel (registered trademark, manufactured by Corning Incorporated) were dissolved in PBS(-), and the solution was filtered through a filter with an average pore size of 0.2 μm to prepare a second solution containing 2% Tetra-PEG-maleimidyl and 1% Matrigel.

Pattern Formation
The substrate was immersed in the first solution and removed, forming a liquid phase of the first solution on the substrate. The second solution was then filled into the liquid chamber of the inkjet head, and the second solution was dropped onto the substrate to form a linear pattern approximately 200 μm wide. As a result, Tetra-PEG-SH contained in the first solution and Tetra-PEG-maleimidyl contained in the second solution crosslinked to form a hydrogel pattern. This hydrogel is a non-cell-adhesive material. Furthermore, the portion coated with Matrigel (registered trademark) formed a pattern of cell-adhesive material. The substrate was then immersed in phosphate-buffered saline (manufactured by Life Technologies, Inc.; hereinafter, also referred to as PBS(-)), and excess first and second solutions were removed.

Preparation of cell ink
Cell ink was prepared in the same manner as in Experimental Example 1, except that human iPS cell-derived GABAergic neurons (manufactured by Elixirgen Scientific) were used as the cells.

<Cell ejection>
Cell ink was filled into the liquid chamber of the cell ejection head of the device shown in Figure 1. Subsequently, droplets of the cell ink were ejected onto the pattern of the cell adhesive material on the substrate, and a liquid pool was formed.

After the cell ink droplets had landed, they were left to stand for approximately 30 minutes, during which time the cells within the droplets settled and temporarily adhered to the substrate, forming cell aggregates. Culture medium was then gently added.

Cell culture
The cells were then cultured in an incubator at 37°C and 5% _CO2 by volume. Figure 13(a) is a bright-field micrograph of cells four days after the start of culture. The scale bar is 100 μm. Figures 13(b) and 13(c) are fluorescent micrographs of Cell Tracker Green fluorescence on the substrate. The scale bar is 100 μm. Figure 13(d) is a fluorescent micrograph of Cell Tracker Green fluorescence in a low cell density region. The scale bar is 50 μm.

The results showed that cells did not migrate to areas where patterns of non-cell-adhesive material were placed. Furthermore, it was revealed that axons were formed only in areas excluding the patterns of non-cell-adhesive material. These results demonstrate that axonal outgrowth of nerve cells can be controlled by placing patterns of non-cell-adhesive material on a substrate.

[Experimental Example 4]
(Non-pattern control of neurons)
A substrate having only the cell adhesive material was prepared in the same manner as in Experimental Example 3. Subsequently, droplets of cell ink were ejected onto the pattern of the cell adhesive material on the substrate, and neurons were seeded in the same manner as in Experimental Example 3. Human iPS cell-derived GABAergic neurons (manufactured by Elixirgen Scientific) were used as the neurons.

The cells were then cultured in an incubator at 37°C and 5% CO2 by volume, and axonal outgrowth of the neurons was confirmed 7 days after the start of culture. Specifically, Calcein-AM (Thermo Fisher Scientific) diluted with _DMSO was added to the medium to a final concentration of 10 μM, and the medium was incubated at 37°C and 5% _CO2 for 30 minutes. The neurons were then observed under a fluorescence microscope.

Figure 14 shows a fluorescence microscope photograph of a neuron. The scale bar is 200 μm. As a result, it was confirmed that when a pattern of non-cell-adhesive material was not placed on the substrate, the direction of neuronal axon outgrowth was not controlled.

[Experimental Example 5]
(Creating a neural circuit model 1)
Neural circuit models were fabricated and evaluated by varying the diameter of the liquid pools on the substrate, the cell density in the liquid pools, the number of cells per liquid pool, the pattern size of the non-cell-adhesive material, and the pattern size of the cell-adhesive material in various combinations as shown in Table 1 below. A 10 cm dish was used as the substrate. Human iPS cell-derived GABAergic neurons (manufactured by Elixirgen Scientific) were used as the neurons.

Figures 15(a) to (e) are schematic diagrams explaining the procedure of this experimental example. Figures 15(a) to (d) are side views, and Figure 15(e) is a top view of Figure 15(b). As shown in Figures 15(a) to (e), the cell adhesive material was arranged in a circular shape to form a cell adhesive region. In addition, a non-cell adhesive material was arranged to surround the cell adhesive region to form a non-cell adhesive region. The shape of the non-cell adhesive region was a roughly donut shape concentric with the adhesive material.

Figure 15(a) is a schematic diagram illustrating the process of placing droplets containing neurons on a substrate. Figure 15(b) is a schematic diagram illustrating the state in which multiple droplets have landed on a substrate, forming a liquid pool. The edge of the liquid pool may be located on the non-cell-adhesive area, but it is preferable that it does not extend beyond the non-cell-adhesive area. Figure 15(c) is a schematic diagram illustrating the state in which neurons in the liquid pool have settled and temporarily adhered to the substrate, forming a cell aggregate. Figure 15(d) is a schematic diagram illustrating the process of supplying a culture medium to the substrate on which the cell aggregate has formed.

In Table 1, "liquid pool diameter" refers to the diameter of the liquid pool placed on the substrate, "cell density" refers to the cell density in the liquid pool, "cell count" refers to the number of cells per liquid pool, "inner diameter/outer diameter of non-adhesive portion" refers to the inner and outer diameters of the donut-shaped portion of the cell non-adhesive portion, "diameter of adhesive portion" refers to the diameter of the cell adhesive portion, and "cell placement method" refers to whether the cells were placed by inkjet (IJ) or manual technique. "Cell density" refers to the number of cells per area where the liquid pool placed on the substrate comes into contact with the cell adhesive portion. "N.D." indicates that measurement was not possible.

The pattern of the non-cell-adhesive material was formed by crosslinking Tetra-PEG-SH contained in the first solution and Tetra-PEG-maleimidyl contained in the second solution, as in Experimental Example 3. To form a pattern of the cell-adhesive material, Matrigel (registered trademark, Corning Inc.) was filled into the liquid chamber of the inkjet head and ejected in a pattern.

The evaluation items for the neural circuit model were the diameter of the liquid pool, the initial cell positioning, and cell fixation. The results of these evaluations are also shown in Table 1.

The goal was to control the diameter of the liquid pool to 500 μm or less, but this was difficult to achieve using manual techniques. However, when placing cells using the inkjet method, it was possible to control the diameter of the liquid pool to either greater than 500 μm or less than 500 μm.

The evaluation criteria for the initial placement of cells were as follows:
-: Cells were randomly arranged.
+: A discrete pattern of cells was formed.

The evaluation criteria for cell fixation were as follows, and cell migration was evaluated on days 1 to 7 of culture.
-: Cells migrated outside the droplets placed on the substrate or were randomly placed.
+: Cells remained inside the droplets placed on the substrate.

As a result, the neural circuit models of Examples 1 to 6 obtained favorable evaluation results in the evaluation items of pool diameter, initial cell placement, and cell fixation. On the other hand, the neural circuit models of Comparative Examples 1 to 6 either had pool diameters exceeding 500 μm or had poor evaluation results in one of the evaluation items.

From the above results, it was revealed that when the droplets placed on the substrate contain seven or more cells per droplet and the cell density in the droplet is ¹⁰⁵ cells/ ^cm2 or more, cells can be stably placed on the substrate even when the diameter of each liquid pool is 500 μm or less.

[Experimental Example 6]
(Creating a neural circuit model 2)
Figures 16(a) to (d) are schematic diagrams showing the arrangement of cells in a neural circuit model of an experimental example in which both a non-cell-adhesive material and a cell-adhesive material were arranged. Figures 16(a) and (b) are side views, and Figures 16(c) and (d) are top views. Figures 16(a) and (c) show the state immediately after the cells were arranged, and Figures 16(b) and (d) show the state after cell culture in which axons have extended between cell aggregates and synapses have been formed.

In this experimental example, the neural circuit model shown in Figures 16(a) to (d) was created. The pattern of the non-cell-adhesive material was formed by crosslinking Tetra-PEG-SH contained in the first solution and Tetra-PEG-maleimidyl contained in the second solution, as in Experimental Example 3. To form the pattern of the cell-adhesive material, Matrigel (registered trademark, Corning Incorporated) was filled into the liquid chamber of the inkjet head and ejected in a pattern. A 10 cm dish was used as the substrate. Human iPS cell-derived GABAergic neurons (Elixirgen Scientific) were used as the neurons. As a result, a neural circuit model was obtained.

[Experimental Example 7]
(Creating a neural circuit model 3)
As shown in Figure 17(a), a neural circuit model was prepared by arranging neurons in the pattern shown in Figure 17(a) on a substrate on which a non-cell-adhesive material was arranged in two concentric circles. The pattern of the non-cell-adhesive material was formed by crosslinking Tetra-PEG-SH contained in the first solution and Tetra-PEG-maleimidyl contained in the second solution, as in Experimental Example 3. Human iPS cell-derived GABAergic neurons (manufactured by Elixirgen Scientific) were used as the neurons.

Figure 17(b) is a representative micrograph of a neural circuit model immediately after cell ink was ejected. In Figure 17(b), "Position: +" indicates that the initial cell positioning was good, and "Position: -" indicates that the initial cell positioning was poor.

After arranging the cells under conditions that allowed for good initial placement, they were cultured for four weeks, allowing axons to extend and resulting in a neural circuit model. Figure 18 shows a fluorescent microscope photograph of the neural circuit model, showing the fluorescence of Cell Tracker Green.

[Experimental Example 8]
(Creating a neural circuit model 4)
As shown in Figures 19(a) and (b), a cell non-adhesive material (Tetra-PEG gel) was placed on a substrate.

The substrate used was a slide glass on which a 13 mm diameter polyester porous culture membrane (trade name: ipCELLCULTURE Track Etched Membrane, pore size: 0.45 μm, pore density: 4 × 10 ⁶ cells/cm ² , thickness: 12 μm, manufactured by it4ip) impregnated with the same first solution as in Experimental Example 3 was laminated.

Then, the ink chamber of the inkjet head was filled with the same second solution as in Experimental Example 3, and the second solution was dropped onto the substrate to form patterns with a width of approximately 200 μm, as shown in Figures 19(a) and (b).

In the pattern shown in Figure 19(a), six linear patterns were formed. The distance between these lines was approximately 0.5 mm. The length of each line was approximately 5 mm. In the pattern shown in Figure 19(b), three concentric circular patterns were formed. The diameters of each pattern, from the center outward, were approximately 1 mm, 2 mm, and 3 mm, respectively.

Next, two types of neurons were arranged in the patterns shown in Figures 19(a) and (b), respectively, to create a neural circuit model. Human iPS cell-derived GABAergic neurons (Elixirgen Scientific) and PC12 cells were used as the neurons.

After arranging the cells under conditions that allowed for good initial placement, they were cultured for one week, allowing neurites to extend and creating a neural circuit model. The neural circuit model was then analyzed by immunostaining.

First, each neural circuit model obtained was washed with PBS and fixed for 30 minutes in 4% paraformaldehyde (Fujifilm Wako Pure Chemical Industries, Ltd.) at 4°C. After fixation, the model was washed once with PBS and blocked for 20 minutes at room temperature using 1% bovine serum albumin (Thermo Fisher Scientific). After blocking, the model was washed once with PBS and a mixture of anti-βIII tubulin antibody (Sigma-Aldrich) and anti-tyrosine hydroxylase antibody (Abcam), each diluted 200-fold with PBS, was added as primary antibodies, and the model was incubated overnight at 4°C.

Then, the sections were washed three times with PBS, and a mixture of APC-labeled goat anti-mouse IgG (H+L) antibody (Thermo Fisher Scientific) and Alexa Fluor 594-labeled goat anti-rabbit IgG (H+L) antibody (Thermo Fisher Scientific), each diluted 500-fold with PBS, was added as secondary antibodies, and the sections were incubated at room temperature for one hour, after which they were washed twice with PBS.

Next, the sample was sealed with a cover glass using sealing liquid (product name "ProLong Diamond Antifade Mountain", Thermo Fisher Scientific), and then observed under a fluorescence microscope.

Figures 20(a) and (b) are fluorescence micrographs of the resulting neural circuit model, observing the fluorescence of βIII tubulin, the neuroskeleton, and tyrosine hydroxylase, which is expressed only in PC12. Figure 20(a) is a fluorescence micrograph of a neural circuit model in which neurons are arranged in the pattern shown in Figure 19(a), and Figure 20(b) is a fluorescence micrograph of a neural circuit model in which neurons are arranged in the pattern shown in Figure 19(b).

As a result, in both neural circuit models in which neurons were arranged in either pattern, βIII tubulin fluorescence was observed throughout the entire cell arrangement area. It was also confirmed that neurites were growing. Furthermore, tyrosine hydroxylase fluorescence was observed only in the area where PC12 cells were arranged, confirming that the cell arrangement pattern was maintained.

[Experimental Example 9]
(Improvement of the drying suppression process)
Cells were seeded on the dry substrate, and the inhibition of drying of the liquid pool and the occurrence of cell adhesion were evaluated. A glass slide was used as the substrate. PC12 cells, a cell line derived from rat pheochromocytoma, were used. The cells were seeded using the inkjet method.

Preparation of cell ink
5 mL of serum-free medium containing green fluorescent dye was added to the cultured PC12 cell dish and cultured for 30 minutes in an incubator (KM-CC17RU2, manufactured by Panasonic Corporation, 37°C, 5% CO2 by _volume environment). The cells were then detached from the dish by trypsin treatment to obtain a cell suspension. Subsequently, a portion of the cell suspension was used to count the number of cells using a NucleoCounter NC-3000 (manufactured by ChemoMetec).

The dispersion medium for the cell ink was PBS(-) supplemented with 0.5% by mass of glycerin (molecular biology grade, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) as a cell drying inhibitor. PC12 cells were dispersed in the dispersion medium to a concentration of 3 x ¹⁰ cells/mL to obtain the cell ink.

<Cell ejection>
The liquid chamber of the cell ejection head of the device shown in Figure 4 was filled with cell ink. Subsequently, droplets of the cell ink were ejected onto the substrate, forming pools. Each pool had a diameter of approximately 400 μm. Each pool on the substrate contained approximately 100 cells, giving a cell density of 8 × ¹⁰ cells/cm ^.

Figure 21(a) is a micrograph of a puddle of cell ink that was covered with biocompatible oil (Oil for Embryo Culture, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) immediately after ejection to prevent drying. By covering the droplet with oil, the tiny puddle did not dry out even when left to stand at 37°C for 60 minutes, and the cells within the droplet settled and temporarily adhered to the substrate, forming a cell aggregate. This method can be applied to many cell types, not just PC12 cells.

The oil was then gently removed and the medium was gently added. Figure 21(b) shows a micrograph taken immediately after the medium was added. Because the liquid pool was a tiny volume, it would dry out in a few minutes in a typical laboratory environment, reducing the volume of the liquid pool by more than 90%.

In addition to methods such as leaving the substrate in a high-humidity environment (Experimental Examples 1-2) and creating a liquid pool on a wet substrate (Experimental Examples 3-8), it was confirmed that covering the liquid pool with oil or the like makes it possible to stably perform cell sedimentation and temporary adhesion even when using a dry substrate.

Next, the number of cells per puddle and the presence or absence of drying-prevention treatment for the puddles on the dry substrate were varied in various combinations as shown in Table 2 below, and neural circuit models of Reference Examples 1 to 4 were fabricated and evaluated. A 35 mm dish was used as the substrate. Human iPS cell-derived GABAergic neurons (manufactured by Elixirgen Scientific) were used as the neurons.

In Table 2 below, "suppression of drying of puddle" indicates whether or not a drying suppression process was performed on the puddle placed on the substrate and the type of process, and "retention of shape of puddle" indicates whether or not the shape of the puddle was maintained after being left to stand at 37°C for 60 minutes. The other items are the same as those shown in Table 1 above. The evaluation criteria for "retention of shape of puddle" were as follows:
+: The shape of the liquid pool was maintained.
-: 80% or more of the liquid pool was dried up and cell death was observed.

The present invention includes the following aspects.
[1] A method for manufacturing a substrate on which neurons are arranged, comprising the steps of: arranging, by an inkjet method, a plurality of droplets containing neurons on a substrate having an area where a cell adhesive material is arranged and an area where a cell non-adhesive material is arranged, to form one or more liquid pools; and allowing the neurons in the liquid pools to settle and temporarily adhere to the substrate to form cell aggregates, wherein the diameter of each liquid pool is 500 μm or less, and the density of neurons per liquid pool is ¹⁰⁵ cells/ ^cm2 or more.
[2] The manufacturing method described in [1], wherein each of the liquid reservoirs contains 7 to 10,000 of the nerve cells.
[3] The manufacturing method described in [1] or [2], wherein each droplet contains 1 to 50 of the nerve cells.
[4] The manufacturing method according to any one of [1] to [3], wherein the droplet is placed in contact with the cell adhesive material.
[5] The manufacturing method according to any one of [1] to [4], further comprising a step of suppressing evaporation of the liquid in the liquid pool.
[6] The manufacturing method described in any one of [1] to [5], further comprising a step of supplying a culture medium to the substrate on which the cell aggregates are formed.
[7] The manufacturing method described in [6] further includes a step of forming multiple liquid pools in the liquid pool forming step, forming multiple cell aggregates in the leaving step, and incubating the substrate supplied with the culture medium to functionally bond at least two of the cell aggregates.
[8] A manufacturing method described in any one of [1] to [7], wherein the substrate has an area where the non-cell-adhesive material is arranged and an area where the non-cell-adhesive material is not arranged, the area where the non-cell-adhesive material is not arranged has a linear shape, and the width of the linear shape is 100 μm or less.
[9] The manufacturing method according to any one of [1] to [8], wherein the substrate has a porous structure.

10...droplet ejection head, 11,970...cell ink, 12,25...liquid chamber, 13,28...membrane, 14,26...drive unit, 15...nozzle, 16,27...vibration unit, 17,24...atmospheric opening unit, 21,22,23...inkjet head, 31...stage unit, droplet placement device...400,500,600,700, 910...flat plate member, 920...porous member, 930...cell culture support, 940...first solution, 950...second solution, 960...non-cell adhesive material, 980...cell, Pj...ejection waveform, Ps...vibration isolation waveform.

Japanese Patent Application Laid-Open No. 2019-162097

Claims (9)

a step of disposing, by an inkjet method, a plurality of droplets containing nerve cells on a substrate having an area where a cell adhesive material is disposed and an area where a cell non-adhesive material is disposed, to form one or a plurality of liquid pools;
and leaving the solution pool to stand until the nerve cells in the solution pool settle and temporarily adhere to the substrate to form a cell aggregate,
The diameter of each of the liquid pools is 100 μm or more and 500 μm or less,
the density of nerve cells per pool is 10 ⁵ to 10 ⁶ cells/cm ² ;
the droplet is placed so as to be in contact with the region where the cell adhesive material is placed, and the density of the nerve cells is the number of cells per area where the droplet is in contact with the region where the cell adhesive material is placed.
A method for manufacturing substrates on which nerve cells are arranged. The manufacturing method described in claim 1, wherein each reservoir contains 7 to 315 of the neurons. The manufacturing method described in claim 1 or 2, wherein each droplet contains 1 to 50 of the neurons. The manufacturing method described in any one of claims 1 to 3, wherein the droplet is placed in contact with the cell adhesive material. The manufacturing method described in any one of claims 1 to 4 further includes a step of suppressing evaporation of the liquid in the liquid pool. The manufacturing method described in any one of claims 1 to 5 further comprises a step of supplying a culture medium to the substrate on which the cell aggregates are formed. forming a plurality of the liquid reservoirs in the step of forming the liquid reservoirs;
forming a plurality of cell aggregates in the standing step;
The method of claim 6 , further comprising the step of incubating the substrate supplied with the culture medium to functionally bond at least two of the cell aggregates. the substrate has an area where the non-cell-adhesive material is disposed and an area where the non-cell-adhesive material is not disposed,
the region where the non-cell-adhesive material is not disposed has a linear shape;
The method according to any one of claims 1 to 7, wherein the width of the linear shape is 100 µm or less. The manufacturing method described in any one of claims 1 to 8, wherein the substrate has a porous structure.